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Electrochemical sensors based on conducting

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Article in Electrochimica Acta · August 2006

DOI: 10.1016/j.electacta.2005.11.052

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 RESEARCH
ARTICLE
Electrochimica Acta (Advanced authors version )
Reviewarticle
Cite this article as: A. Ramanavicius, A. Ramanaviciene, A.
Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–
6037.
Journal version is available at:
http://www.sciencedirect.com/science/article/pii/S0013468606003252

Electrochemical sensors based on

conducting polymer – polypyrrole
(Review)
A. Ramanavicius1,2∗, A. Ramanaviciene1,2, A. Malinauskas3
1.Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, 03225 Vilnius, Lithuania
2.Laboratory of Immunoanalysis and Nanotechnology, Institute of Immunology of Vilnius University, Mol˙et˛u pl. 29,
08409 Vilnius, Lithuaniac
3. Department of Organic Chemistry, Institute of Chemistry, Goˇstauto 9, 01108 Vilnius, Lithuania
∗ Corresponding author. Tel.: +370 5 2330987; fax: +370 5 2330987. E-mail address: arunas@imi.lt (A. Ramanavičius).

DOI:

Abstract
Conducting polymers can be exploited as an excellent tool for the preparation of nanocomposites with
nano-scaled biomolecules. Polypyrrole(Ppy) is one of the most extensively used conducting polymers
in design of bioanalytical sensors. In this review article significant attention ispaid to immobilization
of biologically active molecules within Ppy during electrochemical deposition of this polymer. Such
unique properties ofthis polymer as prevention of some undesirable electrochemical interactions and
facilitation of electron transfer from some redox enzymes are discussed. Recent advances in
application of polypyrrole in immunosensors and DNA sensors are presented. Some new
electrochemical targetDNA and target protein detection methods based on changes of semiconducting
properties of electrochemically generated Ppy doped by affinityagents are introduced. Recent progress
and problems in development of molecularly imprinted polypyrrole are considered.
© 2006 Elsevier Ltd. All rights reserved.
Keywords:Conducting polymers; Polypyrrole; Biosensor; DNA sensor; Immunosensor; Molecularly
imprinted polymers; Bioelectrochemistry; Nanotechnology;Nanobiotechnology
biomolecules,mainly proteins and single stranded DNA
1. Introduction oligomers. Some conductingpolymers doped and/or covalently
or not covalentlymodified by bionanomaterials mentioned
Nanotechnology is rapidly evolving to open new exhibit unique catalytic[1] or affinity [2] properties that can be
materialsuseful in solving challenging bioanalytical problems, easily appliedin the design of bioanalytical sensors
includingspecificity, stability and sensitivity. Here conducting (biosensors).
polymers can be exploited as an excellent tool for the Polypyrrole is one of the most extensively used conducting
preparationof nanocomposites with entrapped nano-scaled polymers in design of bioanalytical sensors [3] as well as for
6025
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
other purposes. Since 1990 up to June 2005 period solelyin the
Journal ElectrochimicaActa over 300 papers appearedon
various properties and applications of polypyrrole.
Versatilityof this polymer is determined by a number of
properties: redox activity [4], ability to form nanowires with
room temperature conductivity ranging from 10−4 to 10−2 S
cm−1 [5], ion-exchange and ion discrimination capacities [6,7],
electrochromic effect depending onelectrochemical
polymerization conditions and charge/discharge processes [8],
strong absorptiveproperties towards gases [9],proteins [10],
DNA [11], catalytic activity [12,13,14], corrosion protection
properties [15], etc. Most of these properties are depending on
the synthesis procedure as well as on the dopant nature
[2].Polypyrrole might be electrochemically generated and
deposited on the conducting surfaces. This technique is
successfully exploited for development of various types of
electrochemical sensors and biosensors. Here several major
directions are straightforward: (i) catalytic sensors based on
immobilized enzymes [1,16,17]; (ii) immunosensors based on
immobilized affinity exhibiting proteins [18]; (iii) DNA
sensors based on covalently immobilized and/or entrapped
ssDNA[19,20,21]; (iv) affinity sensors based on molecularly
imprinted polymers [22].Versatility of this polymer is
determined by the following: its biocompatibility; capability to
transduce energy arising from interaction of analyte and
analyte-recognizing-site into electrical signals that are easily
monitored; capability to protect electrodes from interfering
materials; easy ways for electrochemical deposition on the
surface of any type of electrodes. Nowadays this polymer
becomes one of the major tools for nanobiotechnological
applications [23].
The aim of this study is to review major advances and
applications of this polymer in design of catalytical
biosensors,immunosensors, DNA sensors and molecularly
imprinted polymerbased sensors.
2. Experimental
2.1.Electrochemical polymerization of polypyrrole versus
chemical polymerization
Polypyrrole was firstly synthesized in 1912 [24]. Polypyrrole
synthesized by conventional chemical methods is insoluble in
common solvents because of strong inter-chain interactions
[25]. Two major ways are applied for polypyrrolesynthesis
which are based on induction of polymerization by different
factors: (i) chemical initiation by oxidative agents [24,26]; (ii)
photo induced synthesis [27]; (iii) electrochemical activation
by anodic current [28]. All polymerization initiation methods
mentioned have particular application, e.g. chemical initiation
by oxidative agents might be successfully applied if a great
amount of polypyrrole is needed for application in the design
of chromatography columns [29] or for some other
purposes.By using chemical [26] or even biochemical
[30]methods itis easy to prepare Ppy particles of different
and/or controlled size ranging from several nanometers up to
several micrometers and/or containing various inclusions.
Moreover, by chemical methods it is possible to uniformly
perform overoxidation of this polymer, what is on special
interest of affinity chromatography since molecularly imprinted
Ppy might be produced, which might exhibit selectivity to
molecules ranging from the small organics [31,32,33] to high
molecular weight biomolecules [22]. Photo induced Ppy
6026
synthesis is attractive in photolithographicapplication of this
polymer, since it allows alterations in synthesized Ppy
morphology by change of excitation light wave length [34] and
theoretically it might be applied for the design of electronic
chips. However, because of slow light induced polymerization
rate this polymerization type is still not very often applied if
compared with chemical or electrochemical polymerization.
By using chemically induced polymerization the Ppy ismainly
produced in the bulk solution and just some amount
ofsynthesized polypyrrole is covering the surface of
introducedmaterials. It means that chemically induced
polymerization isnot very efficient with respect to deposition of
Ppy over somesurfaces. Moreover, Ppy is almost insoluble in
usual solvents,except some cases where it is doped with proper
agents increasingsolubility of this polymer [35] and it means
that deposition(e.g. by solvent evaporation) of this polymer
from the solutioncontaining dissolved polymer is possible at
the stage where thepolymer is still in the form of colloid
particles, before its precipitation [30].However, the major
obstacle for use of this deposition method for designing of Ppy
based sensors is a poor adherence of this deposit to the surface,
contrary to the film obtained by electrochemical
polymerization. But all these disadvantages might be avoided if
electrochemical polymerization is applied. It allows deposition
of Ppy over electrodes deposited in the electrochemical cell.
That is the reason why electrochemical polymerization has
found an application as a general deposition method if thin Ppy
layers are requested. By using this method thickness and
morphology of deposited layer might be controlled by
application of well-defined potential and known current
passing through the electrochemical cell [36]. Electrochemical
deposition of Ppy might be performed from various solvents
(e.g. acetonitrile, water, etc.). From the point of view of
nanostructuring of this polymer it is really very important that
Ppy synthesis might be performed from water solution at
neutral pH, since it opens the ways for entrapment and/or
doping of polypyrrole by various biomaterials like small
organic molecules, proteins, DNA and even living cells.
However, if buffers with low bufferingcapacitance are used as
polymerization solution, a potentialproblem is the local
production of a great amount of protonsin the course of the
polymerization which may affect the properties of the
biomolecules to be entrapped inside the Ppy film. In particular
cases overoxidizedPpy might be synthesized and entrapped
molecules and/or dopants might be extracted from the Ppy
structure. In such cases so called molecularly imprinted
polymers might be designed. Moreover, electrochemical
polymerization is applied for deposition of polypyrrole layers
inside geometrically complicated electrochemical cells [37]
and there is almost no doubt that this polymerization method
might be extremely useful for deposition of Ppy layers inside
microfluidic devices. Furthermore, electrochemically
synthesized Ppy has some attractive features, such as good
conductivity and very high adherence of these films to the
mostly for biosensor design used substrates leading towards
sufficient stability of biosensors, even in a neutral pH region.
On the other hand, the electrochemical properties of
Ppystrongly depend on the redox state of this polymer. At
positive potentials an overoxidation of Ppy is occurring what is
leading towards lowering of Ppy conductivity and makes easier
leakage of anionic molecules if they were included into
polymeric backbone. Overoxidation of Ppy appears at lover
positive potentials in water and/or oxygen-containing
environment
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
and in this case it is leading towards partial destruction of
polymeric backbone and generation of oxygen-containing
(carboxyl, carbonyl and hydroxyl) groups. OveroxidizedPpy
has been used in many electroanalytical applications that utilize
its permselectivity and is often used as discrimination
membrane which significantly increases selectivity of
electrochemical biosensors [38,39]. The capability of
electrochemical polypyrrole synthesis is significantly extended,
since some different electrochemical techniques might be
applied for deposition of Ppy over the electrodes: constant
potential electrodeposition, galvanostatic deposition, cyclic
voltammetry, and potential pulse techniques [40].
According to our experience based on application of
conducting polymers in biosensor design the potential pulse
technique is the most suitable for nanostructuring of Ppy by
entrapment of biologically active materials within backbone of
this polymer. Potential pulse techniques enable to increase
concentration of entrapped biologically active material within
nano-thin layers of polypyrrole[40] because various potentials
might be applied in step manner. Higher potential steps are
applied for initiation of Ppy polymerization and lower or
negative potential steps are used for attraction of higher
amount of biomaterial, which is entrapped into polymeric
backbone during polymerization step that is initiated by
potential in the range of +0.6 to +1.2V versus Ag/AgCl. The
number of potential steps, the profile of potential applied and
duration of each step might be set up individually depending on
the application of determined requirements. All factors
mentioned enable to prepare large variation of nanostructured
polymeric layers with different analytical characteristics even
if the same bulk solution is used for polymerization. In general,
electrochemical polymerization is more versatile if compared
with chemical one in terms of possible variations and control of
polymerizationconditions. Moreover, combination of
electrochemical techniqueswith some chemical surface
modification techniquesopens new opportunities for
development of new nanobiostructuralassemblies based on this
polymer. More in detail, it wasdemonstrated that the surface of
electrochemically depositedPpy after some additional
electrochemical/chemical functionalizationmight be covalently
modified by enzymes [41]. Thosestructures were applied in
bio-catalytic biosensor design, andit was demonstrated that Ppy
layers modified by the sameenzyme exhibit significantly
different selectivity towards varioussubstrates if different Ppy
modification approaches areapplied.
However, there are some particular cases where chemical
methods have some advantages if compared to electrochemical
methods. Chemical methods are still mostly used if large extent
of Ppy or appropriate Ppy structures e.g. nanoparticles orPpy
coated nanoparticles of other materials are needed.
Nanocomposites of chemically synthesized polypyrrole are
mainly applied for affinity chromatography purposes [33], but
electrochemical methods are mainly used for construction of
chemical sensors, biosensors and actuators. So, in general, both
Ppy synthesis methods are finding particular application areas
for various technological purposes.
2.2. Polypyrrole as versatile immobilization matrix in
design of biosensors
Most important considerations during the creation of any type
of electrochemical biosensors are: (i) the immobilization of the
bio-catalyst; (ii) application of appropriate electrochemical
technique (e.g. potentiometric, amperometric and impedimetric
techniques are mainly applied for analytical signal registration
in design of electrochemical sensors and biosensors); (iii)
establishment of efficient electron transfer if amperometric
detection is applied. Consequently, immobilization of
biologically active material is of pivotal importance in the
creation of biosensors [42], since it allows application of the
same biologically active material for a number of analysis
cycles. The requirements for successful biomaterial
immobilization are: (i) biological recognition properties and/or
catalytic properties of biomaterial should remain after
immobilization; (ii) the biomaterial should be well fixed
on/within the substrate, otherwise the biosensor will lose its
activity; (iii) improve or at least minimally decrease selectivity
of constructed biosensor or bioanalytical system; (iv) improve
electron transfer if amperometric measurements are applied as
signal transduction system. To solve the majority and
sometimes all these tasks, conducting polymers can be
considered as a very effective substrate for biomaterial
immobilization. Among other conducting polymers,
polyaniline is often used as immobilizing substrate for
biomolecules[39] and sometimes as efficient electrocatalysts.
However, thenecessity to detect bio-analytes at neutral pH
range leads to electro-inactivity of the deposited films,
discouraging the use of polyaniline and polythiophene as
biosensing materials. As opposed to polyaniline, polypyrrole
might be easily deposited from neutral pH aqueous solutions
containing pyrrole monomer. It makes this polymer very
attractive and at present it is one of the most extensively
studied materials useful for immobilization of different
biomolecules and even living cells. On the other hand, Ppy is
often used in catalytic and affinity biosensors because of good
biocompatibility and the easy ways for immobilization of
biomolecules [43]. Biomaterials might be immobilized by
various
methods: (i) adsorption on electrochemically or chemically
formed Ppy surface [44]; (ii) entrapment during
electrochemical deposition of polypyrrole[17,28,36,37,40]; (iii)
self entrapped if biomaterial is able to initiate polypyrrole
synthesis [30]. Asit was mentioned in previous paragraph, by
chemical initiationit is easy to produce large extent of
polypyrrole modified withbiomaterials, however, this method
is not well suitable for theformation of well defined layered
structures such as usually areneeded for sensor design. On the
contrary, the electrochemicalPpy polymerization allows the
formation of uniform films, thethickness and morphology of
such films might be controlled by regulation of passing current
and/or potential applied. Here the application of pulsed
potential techniques allows preconcentration of biologically
active molecules (e.g. DNA, enzymes, etc.) by applying of
proper potential between the pulses initiating polymerization of
polypyrrole[45]. In majority of cases the pulse technique
allows at least to avoid a strong diminution of biologically
active compound concentration near the electrode surface
which takes place at the steady-state diffusion regime and it
strongly enhances amount of inside the film incorporated
biologically active compound if compared to the steady-state
polymerization. Moreover, it was shown that Ppy is able very
effectively discriminate cations and anions, since permeability
and permselectivity of Ppy depends on the counter ion
incorporated during polymerization as well as on the ions
present in the sample [7]. In particular cases anions (e.g.
phosphate) doped electrochemically deposited Ppy if properly
doped by some anions might be not permeable for anions what
6027
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
is very useful for electrochemical biosensors since the majority
of electrochemically interfering materials present in biological
samples are anions [38,39]. It was also demonstrated that Ppy
protects electrodes from fouling by proteins and another
biological substances present in the real samples as blood
serum and urine [46]. It was shown that various
nanocomposites that might be designed using combinatorial
methods by polymerization of a number of electrochemically
polymerizable compounds are useful
for biosensor design [47].
The stability of Ppy based biosensors is sufficient and mainly
determined by degradation of Ppy in the water surrounding if
biosensor is applied for continuous measurements. In
conclusion, from the point of view of electrochemical
biosensingPpy has a number of very attractive characteristics:
(i) it might be synthesized electrochemically and modified by
enzymes in several different ways that gives different
analytical characteristics for constructed biosensors; (ii) it
protects electrodes from fouling and interfering materials such
as electroactive anions; (iii) it is biocompatible and, hence,
causes minimal and reversible disturbance to the working
environment; (iv) in some particular cases it might be exploited
as redox mediator able to transfer electrons from the redox
enzymes towards electrodes.
2.3. Catalytic biosensors based on polypyrrole
Catalytic biosensors are described as compact
analyticaldevices, incorporating a bio-catalytic element or
integratedwithin a transducer system [48]. The detection of
analyte inthis kind of biosensors is based on specific catalytic
conversionof the analyte of interest by a bio-catalyst
immobilized onthe suitable signal transducer. The specific
interaction of analytewith bio-recognition element results in a
change of one ormore physicochemical properties (e.g. electron
transfer, capacity,optical properties) which can be detected and
measured viasignal transduction and registered by registration
devices.Electrochemicalcatalytic biosensors are on special
interest since theycan be applied for detection of analytes in
non-transparent samplesand the majority of electrochemical
catalytic sensors arerelatively cheap and easy in application.
Fig. 1. Generalized scheme of electrochemical biosensors
based on glucoseoxidase. Mox, oxidized form of redox
mediator; Mred, reduced form of redoxmediator.
Thus, as it was mentioned previously, the redox enzymes
aremainly used in the design of catalytic biosensors. This class
6028
ofenzymes can be divided into several major subtypes that
differwith respect to cofactors involved into catalytic reaction.
Cofactorsmainly effect the signal transduction process and
should betaken into account during catalytic biosensor
construction and allthese biosensors have some specific
properties mainly related tothe used enzymes. If enzymes are
applied in the design of amperometric biosensors efficient
electron transfer route is crucial for registration of analytical
signal and the most successful sensorsare so called “reagent
less” biosensors that are operating withoutaddition of any other
soluble materials essential for analyticalsignal registration.
Successful application of polypyrrole modified by enzymesin
the design of catalytic biosensors started by entrapmentof
glucose oxidase from Aspergilusnigerwithin
polypyrrole[49,50], later polyaniline was also successfully
modified withthis enzyme [51,52] and employed for glucose
sensing. Now,FAD-dependent oxidases [53,54,55,56,57],
NAD+-dependent dehydrogenases [58,59,60,61,62], PQQ-
dependent dehydrogenases [16,63,64,65,66,67], peroxidases
[68,69,70,71] and some multicofactor enzymes [72,73,74]are
mostly used in the design of catalytic biosensors. The
embedment of enzymes within a conducting polymer film
prevent the enzyme from being leached out, while at the same
time maintaining accessibility of the catalytic sites due to the
permeability of the film to analytes[75]. Pulse technique for the
electrochemical deposition of polymer films on electrode
surfaces enabled the increase in the concentration of entrapped
enzyme within thin layers of Ppy[40]. Further, the enzyme
activity is usually detected by characterization of final reaction
products or redox mediators using amperometric or
potentiometric methods.
In the case of application of FAD-dependent oxidases
oxidation of enzymatically produced H2O2 is only possible at
high electrode potentials (Fig. 1). Some suitable redox
mediatorsable to facilitate the electron transfer between the
active site of the enzyme and electrode can be applied in the
design of electrochemical catalytic biosensors. So-far described
oxidasesentrapped within conducting polymer-based
biosensors are requiring soluble redox mediators or conducting
polymer backbone should be enhanced by redox species [76].
The most successful biosensors based on oxidases
entrapped within polypyrrole were reported when redox
polymers were constructed on the basis of pyrrole which was
copolymerized with pyrrole substituted by redox mediators.
Thus, for fixing of redox mediators several main strategies
might be applied, e.g.: (i) osmium bipyridine complex based
redox species were attached to pyrrole and after
copolymerization of this compound with pyrrole several
different redox polymers able to transfer electrons from redox
center of glucose oxidase were designed [64,77]; (ii) PQQ-
dependent glucosedehydrogenase was wired by ferrocene
derivatives [78]; (iii) modification of polymeric layers with
some soluble mediators that are almost freely diffusing within
the film [67,63,65,74].
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
Fig. 2. Generalized scheme of electrochemical biosensors
based on NAD+- dependent glucose dehydrogenase.Mox,
oxidized form of redox mediator;Mred,reduced form of redox
mediator
In the case of application of oxidases the sensor response was
dependent on the availability of molecular oxygen; it is a
significant drawback because this concentration is not constant
and might significantly differ from sample to sample. This
drawback might be avoided by application of dehydrogenases
since these enzymes do not require any oxygen as electron
acceptor. Dehydrogenases that are suitable for application in
amperometric biosensors might be divided into two major
subclasses: NAD+-dependent dehydrogenases, and PQQ-
dependent dehydrogenases. In the case of theNAD+-dependent
dehydrogenases (Fig. 2) one may successfully circumvent the
problems imposed by molecular oxygen [79].
Fig. 3. Generalized scheme of electrochemical biosensors
based on PQQdependent glucose dehydrogenase.Mox,
oxidized form of redox mediator;Mred, reduced form of redox
mediator.
However, in the case of NAD+-dependent
dehydrogenaseapplication the coenzyme should be added to the
analyte solutionwhich is only possible in specifically designed
flow-injectionsystems [80], or should be entrapped within
conducting polymerbackbone [81] or graphite paste electrode
matrix [82]. Therefore,intensive attention was focused on
finding new enzymes withimproved characteristics, such as
exhibit the PQQ-dependentdehydrogenases (Fig. 3) [83]. The
use of PQQ-dependentenzymes is more promising, since these
enzymes are oxygen
independent, and their PQQ-cofactor in some cases is tightly
bound within the enzyme’s active site [84]. Significant
advantagewas achieved when polypyrrole based on osmium-
complexmodified redox polymer with entrapped PQQ-
dependent glucosedehydrogenase was applied for glucose
sensing independenton oxygen concentration fluctuations [64].
Some multicofactor enzymes, like PQQ and heme-c
based alcohol dehydrogenase were found to be able to transfer
electrons directly to some conducting surfaces including
polypyrrole (Fig. 4). Such enzymes might be easily applied in
the design of amperometric biosensors based on polypyrrole
because application of any redox mediators is not essential in
this case. It was shown that PQQ-dependent alcohol
dehydrogenase (QH-ADH) covalently attached to the backbone
of polypyrrole retains its catalytic activity [41]. Moreover, it
was demonstrated that QHADH entrapped within polypyrrole
exhibit direct electron transfer to this polymer [72]. It might be
predicted that Ppy and hemec- containing dehydrogenases
based polymeric configurations might be promising in the
design of biofuel cells [85] and other bioelectronic devices
[86]. However, there is just a very limited number of such
enzymes able to directly transfer electrons toward backbone of
conducting polymers. On the other hand, the highest currency
densities in biosensors based on PQQ and
heme-c based alcohol dehyrogenase were achieved when they
were deposited over electropolymerized 4-ferrocenylphenol, N-
(4-hydroxybenzylidene)-4-ferrocenylaniline, and 2-ferrocenyl-
4-nitrophenol [78].
Biosensors with different selectivity and activity
characteristicsmight be designed, since several different
conceptions might be used for immobilization of the same
enzymes: (i) adsorption of enzymes over electrochemically
deposited Ppylayer [44,87,88]; (ii) covalent attachment of
enzymes after introduction of amino groups into Ppy backbone
andformation of amide bounding due to interaction
Fig. 4. Generalized scheme of electrochemical biosensors
based on direct electron transfer able PQQ-dependent alcohol
dehydrogenase. H, heme-c moiety.
with carbodiimide activated carboxylic groups of enzyme
[46,41,89]; here two major different Ppy functionalization
ways might be applied one based on application of amino
group modified pyrrole monomers forcopolymerization with
unmodified pyrrole monomers, next onintroduction of
functional groups following after preparationof Ppy film; (iii)
entrapment within backbone of polypyrrole[17,40,59,64,89].
Entrapment of enzymes within polypyrroleseems the most
promising for construction of catalytic biosensors,since this
method allows to entrap significant amount ofredox enzymes
that are able to convert high amount of analyteinto the products
6029
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
and this process causes high changes ofelectrochemical signals.
In some cases after enzyme entrapmentduring electrochemical
deposition of Ppy over electrodes it wasdetected that enzyme
reactivation phase is very actual before sensormight be applied
for exact analyte determination. In severalcases this phase was
15–30 h long depending on the thickness ofPpy layer formed
[17,74]. The existence of this effect is determinedby swelling
of Ppy because there are some evidencesthat immediately after
the electrochemical deposition enzyme istrapped within very
dense polymeric structure which increasessteric hindrances for
the substrate and deforms native structureof the enzyme. After
some swelling period water permeablecavities become larger
and substrate has more possibilities fordiffusion, as well as
enzyme has more space to become nativeconformation which
possesses highest activity if compared withother—not natural
conformations. It was also demonstrated thateven after
swelling period Ppy is able to accept electrons fromredox
centers of some redox enzymes and transfer those electrons to
the metal electrodes [17]. It seems that polypyrrole is an
inherently biocompatible material. The sufficient water content
ensures that the surface energy of this material is such that it
causes minimum disturbance to the biologically active
compound. In recent years, many catalytic biosensor
configurations and transducers have been designed allowing us
to detect the analyte in very lowconcentrations and with high
precision.However, for practical applications, a few main
problems remain to be solved: (i) for one-way biosensors the
production must be so reproducible that calibration-free
measurements can be performed; (ii) long-term stability in
water containing environment should be increased.
2.4. Immunosensors based on polypyrrole
Immunosensors are the subject of increasing interestmainly
because of their potential application as an
alternativeimmunoassay technique in areas such as clinical
diagnosticsand environmental control. Enzyme-linked
immunosorbentassay (ELISA) is one of the most frequently
used methodsfor immunoassay, because of its good sensitivity,
selectivity,and ease in use. Although spectrometric methods are
widelyused for the detection of enzymatic products resulting
fromthe Ag–Ab reactions in ELISA, the electrochemical
methodscan provide capabilities of monitoring, free from color
and turbid interferences and which are relatively inexpensive,
that the spectrophotometric methods cannot compete with them
[90].Electrochemical affinity sensors acting on the principles
similarto ELISA usually are cheaper and faster in use if
compared totraditional ELISA. Moreover by immunosensors
samples without any analyte enrichment can be analyzed. In
many cases the purification and/or sample pretreatment step is
not needed, which is normally essential for standard analytical
methods such as mass spectrometry, gas chromatography and
high performance liquid chromatography. This factor is
important formany applications, especially in clinical
diagnostics, where differentanalytes in whole blood, serum or
urine containing a lotof different substances, such as proteins,
amino acids, sugars,hormones, etc., are analyzed. Also
immunosensors have considerable advantages over standard
methods with respect to timeand sensitivity [33].
Major indispensable condition during the development
ofaffinity sensors is immobilization of analyte binding
reagent.This task is often solved by application of conducting
polymer,polypyrrole, since Ppy is the mostly used conducting
6030
polymerin affinity sensors because of the best biocompatibility,
due to
efficient polymerization at neutral pH and very easy ways for
immobilization of various biologically active compounds. On
the other hand, it seems that this polymer is capable to transfer
energy as electrochemical transducer [20]. Affinity sensors
mainly rely on immobilized biomolecules or artificially
formedstructures able to interact non-covalently with analyte as
interactionpartner and to form multimolecular complexes.
Amongsuch non-covalently interacting materials are DNA,
antibodies,various proteins and molecularly imprinted
polymers. Thedevelopment of immunosensors would lead to
alternatives orat least improvement in the existing
immunoassay techniques.Most related methods to
immunoassay are immunosensors; theyare mainly applied in
areas where both high selectivity andhigh sensitivity are
required [91]. Immunosensor is a devicethat is able to detect
the interaction between an antibody (Ab)and an antigen (Ag)
[92]. One of the binding able materials inimmunosensors is
usually immobilized and at least one must befound in sample
as analyte. The conversion of the binding eventinto a
measurable signal, the regenerability and the reusabilityare
among major topics and challenges in
immunosensordevelopment research. The conducting polymers
and especiallypolypyrrole can be considered as effective
material for immobilization of biomaterials and for
transducing/amplification ofanalytical signal in design of
immunosensing devices [72,93].Electrochemical modification
of electrodes by conducting polymersdoped with biologically
active compound included withinpolymeric backbone is a
simple step that is often used for creationof different
immunosensors.
In the design of immunosensors antibodies [94,95],
ligands/receptors [96] and antigens [130] are applied as
biologicalmaterials able non-covalently to bind analyte.
Antibodies areconsidered to be well-suited and mostly used
recognition elementsfor construction of immunosensors. The
high specificityand affinity of an antibody for corresponding
antigen allowsa selective binding of the analyte which is
present in nanotopico-molar range in the presence of hundreds
of other substances,even if they exceed the analyte
concentration by 2–3 orders of magnitude [97]. At the time,
antibodies can be generatedagainst almost all analytes, even if
the analyte is nonimmunogenic.Moreover, recombinant
antibody technology has now been developed to the level that
allows the expression of single chain fragments in E. coli in
large quantities [98].In many designs of electrochemical
immunosensors secondaryantibodies able to recognize analyte
complex with immobilizedreceptor were applied. Since
antibodies are usuallynot electrochemically active within the
desired potential rangeredox-active compounds and/or
enzymes (mainly horseradishperoxidase) [99,100,101,102] that
are able to generate electrochemicalsignal can be applied as
labels for indication. Labeledimmunosensor format belongs to
indirect analytical signal detectionmethods. In indirect
electrochemical immunoassays thebinding reaction is
visualized indirectly via an auxiliary reactionby a labeling
compound. Amperometric transducers in
indirectelectrochemical immunoassay are used much more
frequentlythan others. For amperometric immunoassay the
labeling redoxcompound should have the following properties:
it should bereversibly electroactive; it should not cause
electrode fouling;chemical groups for coupling should be
available [103].Species such as nitrophenol, H2O2, and NH3
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
that can be determinedelectrochemically are the substrates or
enzymatic reactionproducts of alkaline phosphatase,
horseradish peroxidase, andurease, generally labeled on
immunoreagents. Among these,because amonia is
electrochemically inactive at low potentialsand can only be
detected by an ammonia gas-sensing electrode,potentiometry is
the only choice for the urease-labeledimmunoassay [104].
Indirect electrochemical immunoassay canbe divided into two
major types: non-amplified and amplified.In non-amplified
redox-labeled electrochemical immunoassaysthe indication of
one antigen or antibody molecule will generateone signal
equivalent. Since the sensitivity of an amperometricsensor for
the redox compound is in the lower micromolar range,this kind
of assay makes sense only if the concentration of theanalyte to
be determined is also in that range [105]. For more
sensitiveimmunoassays amplification principles are necessary.
Oneway to amplify the amperometrical signal is
preconcentrationstep. During this step concentration of redox-
active compoundis increasing many times and only after some
time (1–5 min) themeasurement starts. However, there are
often some difficultiesto regenerate the sensor before each
measurement.
The major disadvantage of indirect immunosensors is the
necessity to apply additional immunochemicals labeled by
electrochemical labels; it makes this method more expensive,
time consuming since additional procedures mainly based on
incubation with labeled antibodies are essential for indirect
detection of analyte of interest. On the one hand, such
procedures increase sensitivity, but on the other hand they
often decrease selectivity since usually broad-range-selectivity
exhibiting secondary antibodies are applied. In this respect so
called ‘label-free’ immunosensors are more attractive, since
such sensors allow measurement without any additional
hazardous reagents evenin real-time [48]. Label-free
conversion of the binding event into a measurable signal in
particular at a low concentration of the analyte, is one of the
major challenges in biosensorics[48]. This topic is quite well
solved in surface plasmon resonance sensors what was
reviewed previously [18]. Electrochemical label-free analyte
detection methods are developing not so fast but they are very
useful if colored and/or not transparent samples are under
investigation or detection of analyte should be performed in the
body of patient. The majority of label-free electrochemical
immunoassays are based on changes in charge densities or
conductivities for transduction and do not need any auxiliary
electrochemical reaction. If conducting polymers are applied
for immobilization of affinity towards analyte exhibiting
reagents after formation of immobilized receptor and analyte
complex changes in capacitance/resistance are registered [106].
Here potentiometric [107], capacitive [102] and
amperometric[99,100,101] transducers have been used for
electrochemical
immunoassays that indicate the binding of analyte directly.
Amperometric techniques have been used to monitor bindingof
analyte in real-time without using a labeled compound.
Apolymer-modified antibody electrode has been used in
combinationwith pulsed amperometric detection. The current
obtainedat the immunochemical/polypyrrole based electrodes
occurs viathe following steps: diffusion of ions to the
electrode; chargetransfer at the porous polypyrrole membrane
interface; migrationthrough the polymer membrane;
adsorption–desorption ofthe analyte at the
immunochemical/polypyrrole interface withsolution. The slow
rate of adsorption–desorption process in thelast step is
considered to be the rate-determining step. Thisstep can be
controlled through the appropriate choice of electricalpotential
[108]. Pulsed amperometric detection (PAD)immunoassay
techniques are such techniques where sensor canbe used for
analyte detection in static or flow injection mode byapplying
pulsed potentials between the sensor surface (or
workingelectrode) and the reference electrode. The current
obtainedcan be directly related to the concentration of the
analyte insolution [109].
Besides amperometric transducers, capacitive transducers
have been used for the real-time and label-free measurement
ofthe Ag–Ab reaction. They are based on the principle that for
electrolytic capacitors the capacitance depends on the thickness
anddielectric behavior of a polymeric layer before and after
interactionwith analyte[110]. In some particular cases
conductivity measurements as one of transduction principle
might be appliedin the design of electrochemical
immunosensors. Conductivitymeasurements have been adapted
for immunoassay based onion concentration increased by the
action of enzymatic label.An enzyme immunoassay based on
conductivity measurementshas been reported, in which urease
was used as the secondaryantibody label. The enzyme retains
the activity under conditionsof lowionic strength, so a
lowbackground conductance could be
employed[111]. However, conductivity measurements are
difficultdue to the variable ionic background of clinical
samplesand the relatively small conductivity changes that are
observedin such high ionic strength solutions. The second
comparative‘blank’ electrode must be used, but variable drift at
two separateelectrodes poses a universal drawback [112].
The inherent speed, accuracy and precision of
electrochemicalmeasurements have stimulated efforts towards
the developmentof both competitive and non-competitive
electrochemicalimmunoassay formats [113]. Sensors
employing enzyme labelswith amperometric detection have
been frequently reported withdrugs[114], hormones
[115,116,117,118] and proteins [119] as targetanalytes, and the
detection of trace amounts in the sub-attomole(<10–18 mol/l)
range has been achieved [120]. In such detectionscheme, the
enzyme label is registered via formed/degraded
electrochemically active product [121], and the function
ofenzyme-labeled immunosensors is similar to that of catalytic
biosensors based on enzymes covalently attached to the
surfaceof polypyrrole. Glucose oxidase, horseradish
peroxidase,microperoxidase, _-galactosidase, alkaline
phosphatase andglucose-6-phosphatase dehydrogenase, all
have been employedin this mode with separation of free from
bound label [122,123,124].The NADH generated by glucose-6-
phophatase dehydrogenasereaction can be readily oxidized by
mediators such as 2,6-dichlorphenolindophenol[125] and 1,4-
benziquinone [126], theoxidation of those is followed
amperometrically
Several immunosensors were applied for continuous
measurements [106,110,127]. The major problem to use the
immunosensor for continuous measurements is stability of
Ag–Ab complexes. To overcome this problem for dissociation
of Ag–Ab complex buffers with extreme pH values, glycine
buffers or extreme salt concentrations are usually used. Flow
injection mode applied together with pulsed-amerometric
detection immunoassay techniques is among such techniques
which can be successfully applied for continuous
measurements in flow throw electrochemical cell
[106,110,127]. Electrochemical label free immunosensors
based on polypyrrole were developed [106,128]. In a novel
6031
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
sampling strategy, antibody-based electrodes were used for the
repeat, intermittent on-line monitoring of tissue corticosteroids
in experimental animals. Here, in a competitive assay, sample
steroid competed with enzyme-labeled corticosteroid for the
antibody immobilized on a platinum electrode surface.
Amperometric detection of the enzyme product was used to
follow the reaction, and to measure enzyme activity related to
the analyte concentration in the sample [129]. However, for
continuous or quasi-continuous measurements of an analyte the
problem of regeneration should be solved, because extreme pH
values, extreme salt concentrations or other factors which are
usually used for dissociation of Ag–Ab complexes lead to
destruction of polymeric immobilization matrix or distortion of
sequence analytical signals. The most immediate potential
applications of immunosensors are medical diagnostics
including the determination of infections [130,131],
environmental analysis, food and beverages control.
2.4. Polypyrrole in the design of DNA sensors
DNA is a unique biomolecule, which is served in known
living species as genetic information storage. Number of
genome projects is providing massive amounts of genetic
information that should revolutionize the understanding of
living nature. Because genome variations between species and
some groups of individuals are straightforward and might be
clearly distinguished it makes specific DNA sequences very
attractive as universal analyte. Nowadays the detection of
appropriated DNA sequences is important for diagnosis of
genetic or infectious diseases, environmental testing for
bacterial contamination, rapid detection of biological warfare
agents, forensic investigations and scientific explorations in
genomics and proteomics.
Moreover, DNA might be determined as important
nanomaterial, which is involved in a number of
nanotechnological and/or bioelectronic applications [132].
There were several demonstrations that DNA in combination
with conducting polymers might be applied for the formation
of unique nanostructures like polypyrrole nanotubes [133] and
polyanilinenanovires[134]. On the other hand, DNA in
combination with polypyrrole was described in a number of
DNA sensors, like in the case of immunosensors,
electrochemically labeled and label-free DNA sensors are
designed. Early works on electrochemical DNA sensors were
mainly based on the application of electrochemical labels for
indication of immobilized single-stranded DNA (ssDNA)
interaction with target DNA present in the sample [135,136].
Such approaches were mainly based on measuring changes in
the peak currents of redox labels, if the DNA duplex formed
during hybridization is exposed to the solution of the indicator
[137]. For this purpose cationic methal complexes such as
tris(2,2_-bipyridine)ruthenium (III) ([Ru(bpy)3]3+) [138] or
tris(1,10-phenanthroline)cobalt(III) ([Co(phen)3]3+) [139],
aromatic compounds such as dye Hoechst 33258 [140],
daunomycin[141] and naphthalene diimine with
covalentlyattached two fereocene moieties [142] or enzymatic
labels (e.g.horseradish peroxidase) based redox indicators
[143]might be applied.
However, the most advantageous are label-free
DNAsensors. Such systems are mainly based on monitoring
changesin electronic or interfacial properties accompanying
DNAhybridization [19]. It means that the key factor
concerningthe development of such electrochemical label-free
DNAsensors is the achievement of efficient interface between
6032
thenucleic acid system and the electronic transducer.
Conductingpolymer molecular interfaces are particularly
suitable formodulating DNA interactions, for inducing
electrical signalsaccrued from such interactions, and for
localizing DNA probesonto extremely small surfaces [19].
Among other conductingpolymers polypyrrole is most
frequently used in the design ofelectrochemical DNA sensors
mainly for immobilization ofssDNA[144]. As it was
demonstrated in several studies, dopingof Ppy by ssDNA is a
very simple procedure if electrochemicaldeposition of this
polymer in the presence of short ssDNAoligonucleotides is
applied [145]. In this way the formedpolymeric layer exhibits
high affinity to complementary ssDNAstrands. Ppy/DNA
sensors are based on various ssDNA immobilization strategies
such as adsorption [11], direct covalentbinding [146],
entrapment in a polymer matrix [20,21,137,147]or indirect
binding by the use of intermediate systems likebiotine-avidine
clips [148]. The most distinct electrochemicalsignals are
generated after DNA hybridization. Two differentways of
ssDNA entrapment might be distinguished: (i) entrapment
in the presence of other counter ions [21,144,147,149];(ii)
doping of Ppy film by ssDNA if ssDNA is present assingle ion
in electrochemical-polymerization solution and isserved as
counter ion [20,153]. In this way polypyrrole bearing
[21,144,147] and doped [20,153] with single-stranded DNA
was applied for the development of DNA sensors. On the
otherhand, polypyrrole is capable to transfer energy as an
electrochemicaltransducer and direct label-free, electrical
detection ofDNA hybridization has also been accomplished by
monitoringchanges in the impedance and capacity/resistance of
conductingpolymer molecular interfaces [21]. Electrochemical
impedance technique seems most informative in label-free
DNA detectionsince this method brings the highest number of
information ontarget DNA interaction with immobilized
ssDNA[150,151].Significant differences in impedance between
electrochemical
systems containing single-stranded DNA immobilizedwithin
polypyrrole and double-stranded DNA (dsDNA) canbe
converted into electrical signals that are easily monitored[152].
However, this method can be successfully replaced byother
less sophisticated electrochemical techniques like
cyclicvotammetry[144], pulsed amperometric detection [21] or
basicamperometry[153]. Garnier et al. have reported a study in
whichnucleic acid probes were linked to the polypyrrole
surface andcyclic voltammetry investigations were performed.
This studydemonstrated a potential shift and wave broadening
in the cyclicvoltammograms registered after interaction with
target DNA[144]. Cyclic voltammetry was applied for
biosensing of DNAhybridization by polypyrrole functionalized
with ferrocenylgroups [154]. Our group has showed that pulsed
amperometricdetection might be applied for detection of target
DNA if ssDNAentrapped within Ppy is deposited over working
electrode [21].Investigations of Wang’s group illustrated that
short termcurrent peaks provoked by the hybridization event at
constantpotential might be registered if short complementary
poly-A,poly-G, poly-T and poly-C oligonucleotides are
interacting with
complementary ones. However, if not-complementary DNA
ispresent in the sample the distinctly opposite current peaks
wereobserved [153]. High stability of Ppy/DNA films allows
detectionof target DNA in flow-trough systems what is a
significantadvantage for continuous routine measurements
[155]. On theother hand, itwas demonstrated that pyrrole–
DNAmight be easilyaddressed towards appropriated electrode
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
what is extremelyuseful for designing electrochemical-DNA
arrays [156].
In some polypyrrole based DNA sensors, quartz crystal
microbalances or fluorescence methods were successfully
applied and such sensors were used for multiple determination
of targetDNA[148]. It might be predicted that newDNAsensors
will be developed where intrinsic electron transfer properties of
DNA[157] and conducting polymers will be combined
together. Next promising direction which is offered by
nanotechnology is application of DNA in combination with
nanoparticles [158]. Here polypyrrole based nanoparticles [30]
might be applied, since previously described DNA sorption on
polypyrrole-silica nanocomposites was very efficient [159].
Carbon nanotubes modified with Ppy/ssDNA seem very
promising for electrochemical DNA sensor design
[150,151,160].
2.6. Application of polypyrrole in molecular imprinting
technology
Indispensable condition during the development of reviously
described affinity sensors is that the bind-able reagents should
be immobilized. However, given to poor chemical and physical
stability of biomolecules even if they are well immobilized,
molecularly imprinted polymers and artificial receptor based
sensors have been gaining importance as a possible alternative
to other affinity sensors (e.g. immunosensors, DNA sensors,
etc.) which are based on immobilized biomolecules [161]. The
preparation of molecularly imprinted polymers requires
polymerization around a print species using monomers that are
selected for their capacity to form specific and definable
interactions with the print species. Within polymer entrapped
molecules can be removed by solvent extraction and the
molecularly imprinted polymer is ready for use. Cavities are
formed in the polymer matrix with ‘images’ of the size and
shape of the imprinted molecules. Furthermore, chemical
functionalities of the monomer residues become spatially
positioned around the cavity in accord with complementarity to
the chemical structure of the imprint molecule. Molecularly
imprinted polymers are very promising during the development
of synthetic recognition systems and are of great interest to
workers in the field of sensor technology. Molecular imprinting
is increasingly becoming recognized as a versatile technique
for the preparation of artificial receptors based on molecularly
imprinted conducting polymers (MIPs) containing tailor-made
recognition sites. MIP is anotherclass of substances of great
interest in the field of chemical sensortechnology[162].
Moreover, these sensors are able to detect lowmolecular mass
organic molecules [31,32,33,163,164]. It is the reason why the
development of synthetic recognition systems is of great
interest to workers in the field of sensor technology [165].
These highly stable synthetic polymers possess molecular
recognition properties due to cavities formed in the polymer
matrix that are complementary to the analyte (ligand) both
inshape and in positioning of functional groups [166]. Some of
these polymers have shown very high selectivity and affinity
constants fully comparable to natural recognition systems
suchas antibodies [167,168,169]. In general, molecular
imprinting is atechnology for the manufacture of synthetic
polymers with predetermined molecular recognition properties
[170]. The preparation of molecularly imprinted polymers
requires polymerization around the print species using
monomers that are selected for their capacity to form specific
and definable interactions with the imprinted species [171].
Furthermore, chemical functionalities of the monomer residues
become spatially positioned around the cavity in a pattern
which is complementary to the chemical structure of the print
molecule [172]. These imprints constitute a permanent memory
for the print species and enable theimprinted polymer to rebind
the print molecule from a mixtureof closely related compounds
selectively [173]. Finally, the printmolecules are removed by
solvent extraction and the molecularlyimprinted polymer is
ready for use. Some of these polymers havebeen shown to be
useful in sensor applications, exhibiting tolerancetowards acid,
base, high temperature and organic phases[174]. It was found
that the manufacture of composites consistingof molecularly
imprinted conducting polymers resultsin obtaining materials
that exhibit both predetermined selectivemolecular recognition
and electrical conductivity [175]. Thistype of materials is of
special interest for use in the field ofsensor technology [176].
Here overoxidizedpolypyrrole is most frequently applied.
Overoxidizedpolypyrrole exhibits an improved
selectivity,which is attributed to the removal of positive
charges from Ppy films due to introduction of oxygen
functionality, such as carbonyl groups. The preparation of
molecularly imprinted polypyrrole requires polymerization
around printed species. Then within Ppy entrapped molecules
are removed by solvent and the molecularly imprinted polymer
is ready for use [29]. Such polymer possesses nano-pores and
nano-cavities that are complementary to removed dopant.
Furthermore, chemical functionalities of the monomer residues
become spatially positioned around the cavity in a pattern that
is complementary to the chemical structure of the print
molecule. Theseimprints constitute a permanent memory for
the print species and enable the imprinted polymer to
selectively rebind the print molecule from a mixture of closely
related compounds. Sensors based on molecularly imprinted
Ppy for serotonin and1-naphthalensulfonate [177], amino acids
[31,175], caffeine [32,163], atropine [178], sacharide[179],
glycoproteins [22] were reported. Both chemically and
electrochemically synthesized Ppy can be applied in the
development of molecularly imprinted polymers. The
electrochemical properties of Ppy strongly depend on their
redox state. At positive potentials overoxidation of polypyrrole
occurs. It leads to the partial degradation of polypyrrole
polymeric backbone and introduction of carboxylic, carbonilic
and hydroxilic groups into polymeric backbone that determines
semi-permeability as well as ability to recognize imprinted
molecules [38]. The best results are achieved if during
electrochemical deposition overoxidizedPpy is imprinted by
small molecular weight molecules [31,32,33,176]. Moreover,
attempts to imprint Ppy by large molecular weight rigid
structure possessing proteins were reported as well as, in this
case viral envelope proteins possessing rigid structure were
imprinted within overoxidizedpolypyrrole[22].
The conversion of the binding event into a measurable
signal in particular at a low concentration of the analyte, the
prevention or elimination of non-specific interactions, the
regenerability, and reusability among other topics are the major
challenges in molecularly imprinted polymer based biosensors.
In such sensors differences in capacitance and/or resistance
arising in electrochemical system during interaction of affinity
agents can be converted into signals that are easily monitored
[180]. Here pulsedamperometric detection can be applied as
basic electrochemical detection method [21,22].
6033
A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors based on conducting
polymer – polypyrrole (Review) Electrochimica Acta 2006, 51, 6025–6037.
3. Conclusions and future trends
Polypyrrole is the most extensively used among other
conducting polymers for the construction of different types of
bioanalytical sensors. The background presented illustrates that
polypyrrole is a very attractive, versatile material, suitable for
preparation of various catalytic and affinity sensors and
biosensors. Both electrochemically and chemically induced
pyrrole polymerization methods has potential application in the
development of analyte-recognizing/converting layers.
In several studies it was shown that Ppy is able to transduce
analytical signal generated by some redox enzymes directly if
redox center of enzyme is deeply buried in the protein globule.
Polypyrrole modified by covalently attached redox groups
might be applied to facilitate electron transfer. From the other
side, the presented overview shows that polypyrrole might be
applied as immobilization matrix in the design of various
affinity sensors like immunosensors, DNA sensors and sensors
based on molecularly imprinted polymers. The use of
polypyrrole in conjunctionwith bioaffinity reagents has
provident to be a powerfulroute that has expanded the range of
applications of electrochemicaldetection and its future
development is expected tocontinue. The interaction between
the proteins, mainly negativelycharged at neutral pH, and the
delocalized positive chargesalong the polypyrrole chains
induces changes in capacitance ofthis nanostructured material.
Consequently, such interactions,evidenced from
electrochemical measurement are the basis ofbioaffinity signal.
The use of a wide range of counterions willprovide significant
change in affinity at the Ppy ion-exchangesites. The application
of nanoelectrode (e.g. carbon nanotubes)technologies already
established in “electronic-nose” deviceswill be beneficial to
polypyrrole based immunosensors. Furtherexploitation of this
technology to immobilize bioaffinityreagents with the polymer
matrix may enable the design ofsmaller, more compact and
portable biosensing systems.
Current achievements show that electrochemical affinity
sensor based on molecularly imprinted polypyrrole could have
a great potential for direct electrochemical sensing. It is straight
forward that in the future molecularly imprinted polymer based
sensors will require deliberate control of the molecular
structure at the surface of the electrode to exhibit higher
affinity to analyte.As the surface microstructure becomes more
complicated, more chemical methods of molecular structure
construction will be required. These methods will use
“molecular technology” instead of bulk technology mainly
used at present time. In addition, for the construction of more
complex nanostructures, somedegree of molecular self-
assembly will be needed and conductingpolymers become
more complex, versatile and will find thenew applications.
New nanotechnological approaches to overcome these
challenges are still in their infancy and application
ofconducting polymers, in particular cases polypyrrole, will
findproper place in future molecular technology.
Acknowledgments
This work was partially financially supported by Lithuanian
State Science and Studies Foundation project number C 03047
and COST program D33.
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